The cells incubated with CHX for 6 h showed mini mal p21 due to the relatively short half life of this protein, a fact which is not altered by the continuing presence of PTEN shRNA and its effects Ruxolitinib msds on PTEN. Inhibition of PI3K or Akt activation reverts p21 stability in PTEN knockdown cells While PTEN acts primarily on PI3K through its lipid phos phatase activity, it is conceivable that there is Inhibitors,Modulators,Libraries a direct effect of PTEN on p21 Inhibitors,Modulators,Libraries that is independent of the PI3K/Akt path way, similar to the effects on PTEN on cellular migration that are mediated partly or exclusively through the C ter minal domains. To investigate this possibility, we tested p21 stability under conditions where we inhibited the activity of either PI3K or Akt. First, we confirmed that loss of PTEN had the expected effects on activation of PI3K by evaluating Akt phosphorylation at Ser 473.

The levels of steady state Akt phosphorylation were high both under conditions of serum deprivation and serum stimulation in PTEN knockdown cells, Inhibitors,Modulators,Libraries indicat ing that attenuation of PTEN removed its antagonistic effects on PI3K mediated Akt activation. Conversely, serum deprivation led to a predictable reduction of phos pho Akt in cells transfected with the mutant control shRNA, yet Akt phosphorylation remained responsive to serum stimulation in these cells, as is observed in wild type ACHN cells. This provides evi dence of specificity, and excludes non specific effects of shRNA transfection on PTEN attenuation. We also observed increased levels of phospho Mdm2 in PTEN knockdown cells, which similarly sup ports a functional effect of PTEN knockdown on related signaling pathways.

Next, we verified that the observed effects of PTEN inhibi tion on p21 stability were indeed mediated through induction of PI3K activity. Incubation of wild type cells with the selective PI3K antagonists LY 294,002 or wortmannin for 0. 5 to 3 hr completely abol ished Akt phosphorylation. Examination of cells grown under the previously established conditions of CHX incubation, Inhibitors,Modulators,Libraries and also in the presence of either LY 294,002 or wortmannin, restored the kinetics of p21 degradation in PTEN attenuated cells to the levels seen in wild type cells and control transfectants. that is, the half life Inhibitors,Modulators,Libraries of this protein was no longer significantly increased in the PTEN attenuated cells than it was in the control cells, indicating that the aug mentation of p21 stability in PTEN knockdown cells was mediated through PI3K.

There is evidence to suggest that enhanced stability of p21 is caused by phosphorylation of p21 on Ser146 by Akt. To determine if activated Akt was required for the stabilization http://www.selleckchem.com/products/Tipifarnib(R115777).html of p21 upon PTEN attenuation, we utilized PIA5, one of a new class of phosphatidylinositol ether lipid analogues that prevent Akt activation and can selec tively kill lung and breast cancer cells with elevated Akt activity.